Rice is considered to be the most important food crop in the world as it is the primary source of dietary calories to majority of human beings. To continue providing adequate calories and to improve the quality of rice-based food in the coming years for the growing population of rice-eaters, the rice plant has to be improved in order to increase yields by exploiting hybrid vigor, to withstand unfavorable conditions like pests, diseases, drought, flooding, salinity and extreme temperatures and to incorporate added nutrients. For a majority of these improvements, genes taken from other organisms, commonly known as transgenes, have to be introduced and expressed in rice.
From agronomic point of view, to generate superior rice varieties, multiple genes conferring desirable genetic traits need to be introduced into the same transgenic background. Accomplishing gene pyramiding, however, is not as easy.
In order to ensure stable expression of the transgenes, they have to be fused to specific DNA elements known as promoters, which function as controlling units for the generation of mRNA from genes, the most important stage of gene expression. These sequences are essentially regulatory in nature, in the sense they initiate the process of transcription and may regulate the rate of transcription. A relatively small number of promoters have been commonly used for expressing transgenes in plants. Additionally, the number of promoters which can drive high levels of constitutive expression in both dicot and monocot plants are even fewer. Also, there are just a handful of promoters driving tissue- or organ-specific expression. The current scientific challenge therefore, lies in the identification of novel regulatory elements to introduce greater versatility in terms of attaining desired spatio-temporal mRNA expression patterns.
Studies have shown that using the same promoter to drive the expression of two distinct heterologous genes in the same plant can lead to gene silencing. This problem can, however, be circumvented by using different promoters for each of the heterologous gene. Hence, there is a need for identifying new promoters which can show tissue- and/or development-specific expression, and can be used for transgene expression in various plants. This document provides such information on a new promoter identified and characterized from the rice tungro bacilliform virus (RTBV), isolated and modified from virus infected rice plants growing in fields in West Bengal, India.
The Cauliflower mosaic virus 35S promoter (CaMV 35S) and its derivatives are among the most commonly used promoters for this purpose. The CaMV 35S is active in dicots, but its relative strength is substantially lower in monocots than in dicots. Other dicot promoters have also been used for monocot transformation, but activity tends to be lower for monocot promoters (Wilmink et al., 1995). Several promoters have been identified to drive a high level of transgene expression in monocots, for example, the rice Act1 promoter (McElroy et al., 1991), the rice rbcs promoter (Kyozuka et al., 1993), the maize Ubi1 promoter (Toki et al., 1992; Cornejo et al., 1993) and the rice cytochrome C gene promoter OsCc1 (Jang et al., 2002). Of these, only the CaMV 35S (Terada and Shimamoto, 1990), rice Act1, maize Ubi1 and rice OsCc1 are constitutive in nature. Only a few tissue-specific promoters have been identified in rice. For example, rice seed storage protein glutelin (Gt1) promoter has been used to express transgenes in rice seeds (Ye et al., 2000) and rice Glu-B1 promoter has been used for endosperm-specific expression of soybean ferritin gene (Goto et al., 1999). Some of the other characterized promoters in rice include, the potato pin2 promoter which has been shown to be wound inducible (Xu et al., 1993), the maize Adh1 promoter which is strongly induced in roots under anaerobic conditions (Kyozuka et al., 1991) and the maize pep and rbcS promoters which are mesophyll specific (Matsuoka et al., 1994).
Beachy, Roger N. and Bhattacharyya, Maitrayee have identified and characterized a promoter from RTBV, isolated from Philippines (U.S. Pat. No. 5,824,857). The utility of the promoter was implicated in driving vascular specific expression in transgenic plants. However, the present study deals with the identification and characterization of a promoter fragment from RTBV, isolated from West Bengal, India, which has no significant homology to the previously-characterized RTBV promoter. The DNA sequence of this full-length clone has been deposited in the EMBL Sequence Database and has been assigned the following accession number: AJ314596, but no function to the DNA sequences was assigned earlier. The present invention deals with the identification of new promoters from RTBV (West Bengal isolate) and their use for transgene expression in plants.